Project A: Establish the role of factors regulating PPi/Pi levels, e.g., ANK, NPP1, PHOSPHO1, and TNAP, in root formation and cementogenesis and apply this knowledge to deliver factors locally/systemically to regenerate periodontal tissues, using rodent models of periodontal disease. Results demonstrate the importance of Pi/PPi and SIBLING family genes/proteins during mineralization, highlighting the need for both physiochemical and cellular molecular factors to achieve homeostatic balance required for formation/regeneration of periodontal tissues. 1. Cell, Tissues and Animal Models of Periodontal Disease: a. Animal Models for regeneration of the periodontal apparatus: Previously, we reported factors controlling PPi/Pi levels have significant roles during formation of the periodontium. In a proof of principle experiment using Ibsp KO mice, exhibiting marked periodontal tissue destruction, TNAP lentivirus, intramuscular delivery, rescued the Ibsp KO phenotype, e.g., insertion of PDL into newly formed cementum and increased alveolar bone volume. In other experiments periodontal fenestration defects were created in ANK and ENPP1 KO mice and at 30 days post-surgery we noted increased cementum regeneration in tissues obtained from KO mice. Data to date suggest different effects of osteoclast like cells on wound healing between tissues analyzed from WT and KO mice and further between tooth root and surrounding bone. We are defining the osteoclast profile in a collaboration with Dr. Ozato s lab. These collective results highlight that factors modulating PPi/Pi levels at local periodontal sites promote cementogenesis. Thus, we initiated studies to delivery TNAP to local sites in Ibsp KO mice. Results are very promising (i.e., cementogenesis promoted) and now, with Dr. Nadine Samara, we are designing TNAP targeted to mineralized tissues for local delivery. We are using in vitro models to determine mechanisms by which TNAP activates mineralization and we continue to evaluate scaffold materials for improved local delivery. b. Ank, Enpp1, dKO and Ank, Alpl dKO mice and cells, in vitro: To determine whether effects of ANK and Enpp1 are additive/synergistic and if mechanistic differences exist in how they control cementogenesis, we generated Ank, Enpp1, dKO and Ank, Alpl dKO mice. Histological and microCT results indicate that acellular cementum phenotype exhibited by the Ank, Enpp1 dKO is a composite phenotype of the single KO. Ank, Alpl dKO mice exhibit improved cementogenesis compared to Alpl KOs, further supporting PPi as a key regulator in cementogenesis. Effects on cementum did not parallel effects on bone. Bone defects appeared more severe in Ank KO and dKO mice compared to Enpp1 KO mice, suggesting that ANK and ENPP1 have non-redundant roles in upregulating extracellular PPi. Additionally, although functional acellular cementum was formed in Ank, Alpl dKO mice, alveolar bone volume and mineral density were not improved compared to Alpl KO mice. Ongoing studies, using proteomics, qPCR microarray and RNA seq technologies are focused toward defining protein and gene expression profiles of PDL tissues obtained from Ank and Enpp1 single and dKO mice at various stages of tooth root development. Further, as mentioned in 1a, we are contrasting periodontal wound healing capabilities of Ank vs Enpp1 KO mice. To complement in vivo studies, Ank KO, Enpp1KO and dKO cementoblasts are being subjected to various mineralization conditions, e.g. Pi or PPi. and data suggest differences in gene expression of osteoclast factors/modulators between cell types. In vitro osteoclast assays using bone marrow stromal cells from Ank and Enpp1 single and dKO mice are being conducted to assess osteoclast differentiation and resorption with ANK and/or ENPP1 loss. c. Orthodontic tooth movement: To continued studies with Dr. Wolf, focused on investigate the role of Pi/ PPi in regulating osteoclast/odontoclast distribution, Ank-/- mice were subjected to orthodontic loading. Also, to characterize the effect of a compromised periodontia on tooth movement, we exposed Ibsp-KAE (BSP RGD replaced by KAE), Ibsp KO, and WT mice to orthodontic loading. We are performing micro CT and histological analyses in tissues obtained from these orthodontically treated mice. Project B: Defining the role of extracellular matrix proteins in periodontogenesis with a focus on SIBLINGS and collagen. BSPxOPN: In 2018 (Foster et al. Bone) we concluded that OPN has a non-redundant role regulating mineralization of dentin and bone, influences tissue properties of PDL and pulp, but does not control acellular cementum apposition. As a next step, we generated and are characterizing periodontal tissues obtained from Spp1(OPN) x Ibsp (BSP) dKO mice to determine if loss of OPN expression rescues the Ibsp KO periodontal phenotype. Preliminary micro CT analysis indicate decreased alveolar bone proper volume in dKO vs. WT, yet greater than that of Ibsp KO mice, suggesting additive, yet complex interactions between OPN and BSP in maintaining periodontal homeostasis. Histological analyses revealed defective periodontal complex and increased number of osteoclasts along the alveolar bone in dKO, like Ibsp KO. Thickened alveolar crest was noted as a unique phenotype in dKO mice. An intriguing finding was huge osteoclasts noted in cells from femurs of Spp1 KO mice but not Ibsp KO or dKO cells. In collaboration with Dr. Hanson Fong, TEM imaging will be used to define further the morphology and function of osteoclasts in vivo and in vitro, obtained from these KO mice. Ibsp-KAE: As a step in defining the mechanism of BSP function in cementogenesis, we generated mice where the BSP RGD domain (integrin binding domain) was replaced by a non-functional KAE sequence (Ibsp-KAE). Results from histology and second harmonic generation microscopy, a collaboration with Dr. Ralston, reveal that in contrast to Ibsp KO mice, Ibsp-KAE mice display normal cementogenesis, but disorganized PDL with significantly increased osteoclasts along the alveolar bone, like Ibsp KO mice. Despite increased osteoclasts, micro CT analysis revealed a higher alveolar bone volume in older age Ibsp-KAE mice. In vitro data examining osteoclastogenesis potential of femur macrophages, demonstrated normal maturation of cells obtained from Ibsp-KAE and Ibsp KO comparable to WT mice. Based on these results we hypothesize that the weakened PDL insertion in Ibsp-KAE mice triggers inflammatory cytokines production, which in turn promotes expression of RANKL and/or RANK, resulting in increased osteoclast activity locally. Next, we analyzed the migration properties of Ibsp KO and Ibsp-KAE cementoblasts in vitro. Ibsp-KAE cells migrated slower and at the same rate as Ibsp KO cells, when compared to WT cells providing evidence for loss of the RGD region as the factor mediating weakened PDL properties we noted in vivo. Another unanticipated finding was that Ibsp-KAE mice exhibit an increase in body weight over time vs WT and Ibsp KO mice. As a first step to understand this phenotype we have initiated studies to determine if these mice (using male mice only at this time) exhibit alterations in specific blood markers to include insulin, glucose and triglycerides and in specific tissues associated with fat metabolism, e.g., liver, pancreas, kidney and epididymis. Collagen: In collaboration with Dr. Nan Hatch, a publication was submitted June 2019, highlighting craniofacial and dental defects in tissues obtained from Crtap mice (inactivating mutations in the gene for cartilage-associated protein).